Etiologic Risk Group:
Persons living or visiting in areas with high concentrations of ticks
Persons who have hobbies or work hunting, trapping, gardening, or landscaping
Persons who work in wildlife management or veterinary medicine
*/Disease Information (sort of like the Intro to your Mini Research Write up): Francisella tularensis causes the disease Tularemia which is a zoonotic disease commonly contracted by insect bites and contact with infected animals. This disease can attack various organ systems including the skin, eyes, lymph nodes, and lungs. The symptoms of tularemia vary depending on where the bacteria enters the body. Ulceroglandular tularemia, the most common form of the disease, causes symptoms such as: an ulcer at the site of infection, swollen or painful lymph glands, fever, chills, headache, and exhaustion. Any symptoms will usually begin 2-10 days after infection. If left untreated, Tularemia can lead to pneumonia, pericarditis, meningitis, and osteomyelitis. This disease is highly contagious, can be fatal, and can be potentially used for bio-terrorism. These factors create a need for more efficient drugs. Currently, tularemia is treated with antibiotics such as streptomycin and gentamicin. Since antibiotics can lead to various side effects and possible drug resistance, more efficient drugs are necessary.
Purification Method: Chromatography using a DEAE-Sepharose FF column
Image of protein (PyMol with features delineated and shown separately): *Amino Acid Sequence (paste as text only - not as screenshot or as 'code'): MKLNKITLGILSLSIATTTFATDVNNSKPNDYGTLVKIEQKLFNNANTLKTTTPIKHVVIIFQENNSFDRYFGMYPNAKNPEGEPKFVAKENTPNVNGLTKQLLENNPNTKNPYRLDRNFQPCSQNHEYHQEISSFNGGLMNKFVEHGGHDNDTYKQNCDGQVMGYYDGNTVTALWNYAQNFALNDNTFGTTFGPSTPGALNLVAGANGPAMSPSGNLENIENNYIIDDPNPYYDDCSYGTSKSGDTNTAVAKITDGYNIGHYLTQKGITWGWFQGGFKPTSYSGKTAICDAMSTNKFGVKSRDYIPHHEPFNYWKETSNPHHLAPSDDKYIGSNDQANHQYDISEFWKALDQNNMPAVSYLKAPGYQDGHGGYSNPLDEQEWLVNTINRIQQSKDWDSTAIIIIYDDSDGDYDHVYSPKSQFSDIKGRQGYGPRLPMLVISPYAKANYVDHSLLNQASVLKFIEYNWGIGSVSKYSNDKYSNNILNMFDFNKEQKTLKLILDPKTGLVMDKLN
*length of your protein in Amino Acids: 514
Molecular Weight of your protein in kiloDaltons using the Expasy ProtParam website: 57780.9 kDa
Molar Extinction coefficient of your protein at 280 nm wavelength:
<span style="background-color: #ffffff; color: #626262;">Ext. coefficient 91930
Abs 0.1% (=1 g/l) 1.591, assuming all pairs of Cys residues form cystines
Ext. coefficient 91680
Abs 0.1% (=1 g/l) 1.587, assuming all Cys residues are reduced
</span>
*CDS Gene Sequence (paste as text only):
ATGGACGTGAATAATTCTAAGCCGAACGATTACGGTACCCTGGTTAAAATCGAACAGAAACTGTTTAACAACGCGAATACGCTCAAAACGACCACCCCGATTAAACACGTAGTCATTATCTTCCAGGAGAATAACTCTTTTGATCGTTACTTCGGTATGTACCCGAATGCGAAGAATCCGGAAGGTGAACCAAAGTTTGTTGCGAAAGAAAACACCCCAAATGTTAACGGCCTCACCAAACAGCTGCTCGAAAATAACCCGAACACCAAAAATCCGTACCGCCTGGATCGCAACTTCCAGCCGTGCTCTCAGAACCACGAATACCACCAGGAAATCTCTTCTTTCAATGGTGGCCTGATGAATAAGTTCGTCGAACATGGTGGTCACGACAATGACACCTACAAACAGAATTGCGATGGTCAGGTGATGGGCTACTATGATGGCAATACTGTTACCGCGCTGTGGAATTACGCGCAGAACTTCGCGCTGAATGACAACACCTTTGGTACTACCTTCGGTCCGTCTACCCCGGGTGCCCTGAACCTGGTTGCGGGTGCAAACGGTCCGGCGATGTCTCCGTCTGGCAACCTCGAGAACATCGAAAACAACTACATCATTGACGACCCTAACCCGTATTATGACGACTGCTCTTACGGCACCTCTAAATCTGGCGACACCAACACTGCGGTTGCTAAGATCACGGATGGTTACAACATCGGTCACTACCTCACCCAAAAAGGTATCACTTGGGGTTGGTTTCAAGGCGGCTTCAAACCGACCTCTTACTCTGGTAAGACGGCGATTTGTGACGCCATGTCTACCAACAAGTTCGGTGTTAAATCTCGTGACTATATCCCGCACCACGAGCCTTTCAACTATTGGAAGGAGACCTCCAATCCACACCACCTCGCGCCGAGCGACGACAAATACATTGGTTCTAATGATCAAGCGAATCACCAATACGACATCTCTGAATTCTGGAAGGCGCTCGACCAGAACAACATGCCTGCCGTTTCTTACCTGAAAGCTCCGGGTTACCAGGATGGCCACGGTGGCTACTCCAACCCACTGGACGAACAGGAATGGCTCGTTAATACCATCAACCGTATCCAGCAGTCTAAAGACTGGGACTCTACCGCTATTATCATCATTTACGATGACAGCGACGGTGATTACGATCACGTTTACTCTCCAAAGTCTCAATTCTCTGACATCAAGGGCCGTCAAGGTTATGGTCCGCGTCTGCCGATGCTGGTTATCAGCCCGTATGCAAAAGCTAACTACGTTGACCACTCTCTGCTGAATCAGGCGTCTGTCCTGAAATTCATCGAATACAACTGGGGTATCGGTTCCGTATCTAAATACTCTAACGACAAGTATTCTAACAATATCCTGAATATGTTTGACTTCAACAAAGAGCAAAAAACCCTGAAACTGATCCTGGACCCGAAAACTGGTCTCGTTATGGATAAACTGAACTAA
*GC% Content for gene: 47.77
*CDS Gene Sequence (codon optimized) - copy from output of Primer Design Protocol (paste as text only): FILTERED DNA SEQUENCE TACTTCCAATCCATGGACGTGAATAATTCTAAGCCGAACGATTACGGTACCCTGGTTAAAATCGAACAGAAA CTGTTTAACAACGCGAATACGCTCAAAACGACCACCCCGATTAAACACGTAGTCATTATC TTCCAGGAGAATAACTCTTTTGATCGTTACTTCGGTATGTACCCGAATGCGAAGAATCCG GAAGGTGAACCAAAGTTTGTTGCGAAAGAAAACACCCCAAATGTTAACGGCCTCACCAAA CAGCTGCTCGAAAATAACCCGAACACCAAAAATCCGTACCGCCTGGATCGCAACTTCCAG CCGTGCTCTCAGAACCACGAATACCACCAGGAAATCTCTTCTTTCAATGGTGGCCTGATG AATAAGTTCGTCGAACATGGTGGTCACGACAATGACACCTACAAACAGAATTGCGATGGT CAGGTGATGGGCTACTATGATGGCAATACTGTTACCGCGCTGTGGAATTACGCGCAGAAC TTCGCGCTGAATGACAACACCTTTGGTACTACCTTCGGTCCGTCTACCCCGGGTGCCCTG AACCTGGTTGCGGGTGCAAACGGTCCGGCGATGTCTCCGTCTGGCAACCTCGAGAACATC GAAAACAACTACATCATTGACGACCCTAACCCGTATTATGACGACTGCTCTTACGGCACC TCTAAATCTGGCGACACCAACACTGCGGTTGCTAAGATCACGGATGGTTACAACATCGGT CACTACCTCACCCAAAAAGGTATCACTTGGGGTTGGTTTCAAGGCGGCTTCAAACCGACC TCTTACTCTGGTAAGACGGCGATTTGTGACGCCATGTCTACCAACAAGTTCGGTGTTAAA TCTCGTGACTATATCCCGCACCACGAGCCTTTCAACTATTGGAAGGAGACCTCCAATCCA CACCACCTCGCGCCGAGCGACGACAAATACATTGGTTCTAATGATCAAGCGAATCACCAA TACGACATCTCTGAATTCTGGAAGGCGCTCGACCAGAACAACATGCCTGCCGTTTCTTAC CTGAAAGCTCCGGGTTACCAGGATGGCCACGGTGGCTACTCCAACCCACTGGACGAACAG GAATGGCTCGTTAATACCATCAACCGTATCCAGCAGTCTAAAGACTGGGACTCTACCGCT ATTATCATCATTTACGATGACAGCGACGGTGATTACGATCACGTTTACTCTCCAAAGTCT CAATTCTCTGACATCAAGGGCCGTCAAGGTTATGGTCCGCGTCTGCCGATGCTGGTTATC AGCCCGTATGCAAAAGCTAACTACGTTGACCACTCTCTGCTGAATCAGGCGTCTGTCCTG AAATTCATCGAATACAACTGGGGTATCGGTTCCGTATCTAAATACTCTAACGACAAGTAT TCTAACAATATCCTGAATATGTTTGACTTCAACAAAGAGCAAAAAACCCTGAAACTGATC
CTGGACCCGAAAACTGGTCTCGTTATGGATAAACTGAACTAACAGTAAAGGTGGATA
*GC% Content for gene (codon optimized):47.65
Primer design results for pNIC-Bsa4 cloning (list sequences of all of your ~40 nt long primers):
Primer design results for 'tail' primers (this is just 2 sequences): Forward Primer:TACTTCCAATCCATGGACGTGAATAATTCTAA Reverse Primer:TATCCACCTTTACTGTTAGTTCAGTTTATCCATAA
*NCBI Gene # or RefSeq#: FTN_0090
*Protein ID (NP or XP #) or Wolbachia#: YP_897755.1
*Organism (including strain): Francisella tularensis
Etiologic Risk Group:
Persons living or visiting in areas with high concentrations of ticks
Persons who have hobbies or work hunting, trapping, gardening, or landscaping
Persons who work in wildlife management or veterinary medicine
*/Disease Information (sort of like the Intro to your Mini Research Write up):
Francisella tularensis causes the disease Tularemia which is a zoonotic disease commonly contracted by insect bites and contact with infected animals. This disease can attack various organ systems including the skin, eyes, lymph nodes, and lungs. The symptoms of tularemia vary depending on where the bacteria enters the body. Ulceroglandular tularemia, the most common form of the disease, causes symptoms such as: an ulcer at the site of infection, swollen or painful lymph glands, fever, chills, headache, and exhaustion. Any symptoms will usually begin 2-10 days after infection. If left untreated, Tularemia can lead to pneumonia, pericarditis, meningitis, and osteomyelitis. This disease is highly contagious, can be fatal, and can be potentially used for bio-terrorism. These factors create a need for more efficient drugs. Currently, tularemia is treated with antibiotics such as streptomycin and gentamicin. Since antibiotics can lead to various side effects and possible drug resistance, more efficient drugs are necessary.
Link to TDR Targets page (if present): n/a
Link to Gene Database page (NCBI, EuPath databases -e.g. TryTryp, PlasmoDB, etc - or PATRIC, etc.):
http://www.ncbi.nlm.nih.gov/gene/?term=FTN_0090
Essentiality of this protein: Known to be essential
Is it a monomer or multimer as biological unit? Most Probable Assembly: 2-mer
Complex of proteins?: Complexed with Othrovanadate (2D1G)
Druggable Target (list number or cite evidence from a paper/database showing druggable in another organism): n/a (Could not be found)
*EC#: 3.1.3.2
Link to BRENDA EC# page: http://www.brenda-enzymes.org/php/result_flat.php4?ecno=3.1.3.2
Enzyme Assay information (spectrophotometric, coupled assay ?, reagents):
Phosphatase assay was performed in 20 mM sodium acetate buffer, 1 mM MgCl2 pH 6 (assay buffer), at 37 °C, using as substrate 1 mM pNPP, in a volume of 250 μl. http://www.sciencedirect.com/science/article/pii/S1570963905003699Structure (PDB or Homology model): 2DIG
http://www.rcsb.org/pdb/explore/explore.do?structureId=2D1G
+
Current Inhibitors:
Zn2+, vanadate, tungstate, Tartrate, orthovanadate, molybdate, Hg2+, FeSO4, Cu2+, arsenate, Ag+, (AgNO3)
Expression Information (has it been expressed in bacterial cells): Has been expressed in E. coli Dh5a cells.
http://www.sciencedirect.com/science/article/pii/S1570963905003699
Purification Method: Chromatography using a DEAE-Sepharose FF column
Image of protein (PyMol with features delineated and shown separately):
*Amino Acid Sequence (paste as text only - not as screenshot or as 'code'):
MKLNKITLGILSLSIATTTFATDVNNSKPNDYGTLVKIEQKLFNNANTLKTTTPIKHVVIIFQENNSFDRYFGMYPNAKNPEGEPKFVAKENTPNVNGLTKQLLENNPNTKNPYRLDRNFQPCSQNHEYHQEISSFNGGLMNKFVEHGGHDNDTYKQNCDGQVMGYYDGNTVTALWNYAQNFALNDNTFGTTFGPSTPGALNLVAGANGPAMSPSGNLENIENNYIIDDPNPYYDDCSYGTSKSGDTNTAVAKITDGYNIGHYLTQKGITWGWFQGGFKPTSYSGKTAICDAMSTNKFGVKSRDYIPHHEPFNYWKETSNPHHLAPSDDKYIGSNDQANHQYDISEFWKALDQNNMPAVSYLKAPGYQDGHGGYSNPLDEQEWLVNTINRIQQSKDWDSTAIIIIYDDSDGDYDHVYSPKSQFSDIKGRQGYGPRLPMLVISPYAKANYVDHSLLNQASVLKFIEYNWGIGSVSKYSNDKYSNNILNMFDFNKEQKTLKLILDPKTGLVMDKLN
*length of your protein in Amino Acids: 514
Molecular Weight of your protein in kiloDaltons using the Expasy ProtParam website: 57780.9 kDa
Molar Extinction coefficient of your protein at 280 nm wavelength:
TMpred graph Image (http://www.ch.embnet.org/software/TMPRED_form.html).
2 Possible Transmembrane Regions
*CDS Gene Sequence (paste as text only):
ATGGACGTGAATAATTCTAAGCCGAACGATTACGGTACCCTGGTTAAAATCGAACAGAAACTGTTTAACAACGCGAATACGCTCAAAACGACCACCCCGATTAAACACGTAGTCATTATCTTCCAGGAGAATAACTCTTTTGATCGTTACTTCGGTATGTACCCGAATGCGAAGAATCCGGAAGGTGAACCAAAGTTTGTTGCGAAAGAAAACACCCCAAATGTTAACGGCCTCACCAAACAGCTGCTCGAAAATAACCCGAACACCAAAAATCCGTACCGCCTGGATCGCAACTTCCAGCCGTGCTCTCAGAACCACGAATACCACCAGGAAATCTCTTCTTTCAATGGTGGCCTGATGAATAAGTTCGTCGAACATGGTGGTCACGACAATGACACCTACAAACAGAATTGCGATGGTCAGGTGATGGGCTACTATGATGGCAATACTGTTACCGCGCTGTGGAATTACGCGCAGAACTTCGCGCTGAATGACAACACCTTTGGTACTACCTTCGGTCCGTCTACCCCGGGTGCCCTGAACCTGGTTGCGGGTGCAAACGGTCCGGCGATGTCTCCGTCTGGCAACCTCGAGAACATCGAAAACAACTACATCATTGACGACCCTAACCCGTATTATGACGACTGCTCTTACGGCACCTCTAAATCTGGCGACACCAACACTGCGGTTGCTAAGATCACGGATGGTTACAACATCGGTCACTACCTCACCCAAAAAGGTATCACTTGGGGTTGGTTTCAAGGCGGCTTCAAACCGACCTCTTACTCTGGTAAGACGGCGATTTGTGACGCCATGTCTACCAACAAGTTCGGTGTTAAATCTCGTGACTATATCCCGCACCACGAGCCTTTCAACTATTGGAAGGAGACCTCCAATCCACACCACCTCGCGCCGAGCGACGACAAATACATTGGTTCTAATGATCAAGCGAATCACCAATACGACATCTCTGAATTCTGGAAGGCGCTCGACCAGAACAACATGCCTGCCGTTTCTTACCTGAAAGCTCCGGGTTACCAGGATGGCCACGGTGGCTACTCCAACCCACTGGACGAACAGGAATGGCTCGTTAATACCATCAACCGTATCCAGCAGTCTAAAGACTGGGACTCTACCGCTATTATCATCATTTACGATGACAGCGACGGTGATTACGATCACGTTTACTCTCCAAAGTCTCAATTCTCTGACATCAAGGGCCGTCAAGGTTATGGTCCGCGTCTGCCGATGCTGGTTATCAGCCCGTATGCAAAAGCTAACTACGTTGACCACTCTCTGCTGAATCAGGCGTCTGTCCTGAAATTCATCGAATACAACTGGGGTATCGGTTCCGTATCTAAATACTCTAACGACAAGTATTCTAACAATATCCTGAATATGTTTGACTTCAACAAAGAGCAAAAAACCCTGAAACTGATCCTGGACCCGAAAACTGGTCTCGTTATGGATAAACTGAACTAA
*GC% Content for gene: 47.77
*CDS Gene Sequence (codon optimized) - copy from output of Primer Design Protocol (paste as text only):
FILTERED DNA SEQUENCE
TACTTCCAATCCATGGACGTGAATAATTCTAAGCCGAACGATTACGGTACCCTGGTTAAAATCGAACAGAAA
CTGTTTAACAACGCGAATACGCTCAAAACGACCACCCCGATTAAACACGTAGTCATTATC
TTCCAGGAGAATAACTCTTTTGATCGTTACTTCGGTATGTACCCGAATGCGAAGAATCCG
GAAGGTGAACCAAAGTTTGTTGCGAAAGAAAACACCCCAAATGTTAACGGCCTCACCAAA
CAGCTGCTCGAAAATAACCCGAACACCAAAAATCCGTACCGCCTGGATCGCAACTTCCAG
CCGTGCTCTCAGAACCACGAATACCACCAGGAAATCTCTTCTTTCAATGGTGGCCTGATG
AATAAGTTCGTCGAACATGGTGGTCACGACAATGACACCTACAAACAGAATTGCGATGGT
CAGGTGATGGGCTACTATGATGGCAATACTGTTACCGCGCTGTGGAATTACGCGCAGAAC
TTCGCGCTGAATGACAACACCTTTGGTACTACCTTCGGTCCGTCTACCCCGGGTGCCCTG
AACCTGGTTGCGGGTGCAAACGGTCCGGCGATGTCTCCGTCTGGCAACCTCGAGAACATC
GAAAACAACTACATCATTGACGACCCTAACCCGTATTATGACGACTGCTCTTACGGCACC
TCTAAATCTGGCGACACCAACACTGCGGTTGCTAAGATCACGGATGGTTACAACATCGGT
CACTACCTCACCCAAAAAGGTATCACTTGGGGTTGGTTTCAAGGCGGCTTCAAACCGACC
TCTTACTCTGGTAAGACGGCGATTTGTGACGCCATGTCTACCAACAAGTTCGGTGTTAAA
TCTCGTGACTATATCCCGCACCACGAGCCTTTCAACTATTGGAAGGAGACCTCCAATCCA
CACCACCTCGCGCCGAGCGACGACAAATACATTGGTTCTAATGATCAAGCGAATCACCAA
TACGACATCTCTGAATTCTGGAAGGCGCTCGACCAGAACAACATGCCTGCCGTTTCTTAC
CTGAAAGCTCCGGGTTACCAGGATGGCCACGGTGGCTACTCCAACCCACTGGACGAACAG
GAATGGCTCGTTAATACCATCAACCGTATCCAGCAGTCTAAAGACTGGGACTCTACCGCT
ATTATCATCATTTACGATGACAGCGACGGTGATTACGATCACGTTTACTCTCCAAAGTCT
CAATTCTCTGACATCAAGGGCCGTCAAGGTTATGGTCCGCGTCTGCCGATGCTGGTTATC
AGCCCGTATGCAAAAGCTAACTACGTTGACCACTCTCTGCTGAATCAGGCGTCTGTCCTG
AAATTCATCGAATACAACTGGGGTATCGGTTCCGTATCTAAATACTCTAACGACAAGTAT
TCTAACAATATCCTGAATATGTTTGACTTCAACAAAGAGCAAAAAACCCTGAAACTGATC
CTGGACCCGAAAACTGGTCTCGTTATGGATAAACTGAACTAACAGTAAAGGTGGATA
*GC% Content for gene (codon optimized): 47.65
Primer design results for pNIC-Bsa4 cloning (list sequences of all of your ~40 nt long primers):
DNA Works Output
Primer design results for 'tail' primers (this is just 2 sequences):
Forward Primer: TACTTCCAATCCATGGACGTGAATAATTCTAA
Reverse Primer: TATCCACCTTTACTGTTAGTTCAGTTTATCCATAA